Autophagy is an evolutionarily-conserved process through which eukaryotic cells degrade intracellular organelles and biomolecules in the lysosome. It is the fundamental cellular process important to maintain the balance between synthesis, degradation and recycling of cellular constituents in response to changes in nutrient levels and other cellular environments. Dys-regulation of autophagy has been implicated in aging, innate immunity and many human diseases including cancers, neurodegenerative diseases, and immune disorders. Despite the recent progress, the molecular mechanisms of autophagy remain still poorly understood. The long-term goal of our study is to define the mechanisms of autophagy and understand how this knowledge can be utilized to improve human health. Mechanistic target of rapamycin (mTOR), the master growth regulatory kinase, is a key regulator of autophagy induction, and the mTOR-autophagy pathway represents a viable target in diseases where autophagy is compromised. During the past years of research since our discovery of the ULK1-Atg13-FIP200 complex as a target of mTOR, we discovered that mTOR engages in a broader range of functions in autophagy than previously thought. The objective of this renewal proposal is to define the expanded roles of mTOR in autophagy. The central hypothesis is that mTOR negatively regulates not only early stages of autophagy, such as omegasome/phagophore formation, but also phagophore expansion and autophagosome maturation. We will test our central hypothesis by pursuing three specific aims: First, we will define the pathway through which mTOR regulates omegasome/phagophore formation. We will determine the functions of mTOR-mediated phosphorylations of Atg13 in ULK1 clustering and the omegasome/phagophore formation. We will use cellular systems we developed using TALEN-assisted genome editing technique to monitor endogenous ULK1 puncta in live cells along with phosphorylation site mutant-reconstituted cellular systems. Second, we will determine the mechanism by which the ULK1 complex regulates phagophore expansion. We discovered MCF2L2, a putative guanine nucleotide exchange factor for Rho family GTPases, as a binding protein of Atg13 and a regulator of Atg9 recruitment. We will determine how ULK1 regulates MCF2L2 via phosphorylation. Third, we will determine the roles of mTOR in autophagosome maturation. Our working hypothesis is that mTOR and ULK1 coordinately regulate autophagosome maturation via phosphorylating UVRAG. We will determine the functions of mTOR- and ULK1-mediated phosphorylations of UVRAG in autophagosome maturation and how this regulation is coordinated with the early stages of autophagy processes. The proposed works will define a broad range of functions of mTOR in autophagy, which will be critical for comprehensive understanding of the fundamental cellular process and provide the key information for potential targets to monitor or manipulate specific steps of autophagy.